Treatment of cancer using benzoic acid derivatives

ABSTRACT

The present invention provides a method of treating cancer using benzoic acid derivatives, alone or in combination with standard treatments such as chemotherapy and radiotherapy. Also provided are methods of screening for benzoic derivatives based on their ability to inhibit the enzyme tyrosinase or to bind to and activate PXR/SXR xenobiotic receptors.

This application claims priority under 35 U.S.C. § 119(e) to U.S.provisional application No. 60/538,360, filed Jan. 21, 2004; thecontents of which are hereby incorporated by reference in theirentirety.

GOVERNMENT SUPPORT

This work was supported in part by NIH grant No. ROICA91645. Pursuant tothe terms of that grant, the federal government may have certain rightsto this invention.

FIELD OF THE INVENTION

The present invention relates to the treatment of cancer usingderivatives of p-amino-benzoic acid (PABA). The invention also relatesto the potentiation of radiotherapy, chemotherapy, or a combinationthereof, with PABA derivatives that are agonists of the PXR/SXRxenobiotic receptor. The present invention further relates to a methodof screening to identify lead compounds that agonize the PXR/SXRreceptor.

BACKGROUND OF THE INVENTION

Cancer

Cancer accounts for nearly one-quarter of deaths in the United States,exceeded only by heart disease. In the year 2000, there were 553,091cancer deaths in the US. In 2003, the American Cancer Society estimatesthat this number will increase to approximately 556,500, due to agingand growth of the population. Lung cancer is the most common fatalcancer in men (31%), followed by prostate (10%), and colon & rectum(10%). In women, lung (25%), breast (15%), and colon & rectum (11%) arethe leading sites of cancer death. Among children, leukemia is the mostcommon cancer among children ages 0-14 years and it comprisesapproximately 30% of all childhood cancers and accounts for the mostchildhood deaths. Acute lymphocytic cancer is the most common form ofleukemia in children. It is estimated that 1.33 million new cases ofcancer were diagnosed in 2003 (American Cancer Society, 2003 CancerStatistics Slide Set 2003).

Melanoma. Studies have indicated that nearly 45,000 new cases ofmelanoma are diagnosed each year in the U.S, and approximately 20% ofpatients will die of metastatic disease. Melanomas arise from themalignant conversion of melanocytes, which in turn are derived frommesenchymal neural crest cells. Melanomas undergo melanogenesis, acomplex process that results in the production of melanin. Melanogenesisis initiated by the hydroxylation of L-tyrosine, to formL-dihydroxyphenylalanine (L-DOPA), which is then converted to DOPAchromeby specific melanocyte-associated enzymes, including tyrosinase. Afurther series of oxidation and reduction reactions ultimately convertDOPAchrome to melanin. It has been suggested that melanogenesis mayaccount for the resistance of melanomas to treatment with ionizingradiation and chemotherapy. It has also been suggested that byproductsof melanogenesis are responsible for other adverse effects includingimmunosuppression, fibrosis and mutagenesis.

Current Treatments

Chemotherapy (CT) and radiation therapy (RT), and combinations thereof,remain the leading defenses against cancer, although recent advances inthe field have led to widespread uses of specialized treatments such asangiogenesis inhibitors, biological therapies, including adjuvanttherapy to boost the patient's immune system, antibody therapy, vaccinetherapy, and photodynamic therapy.

In addition to numerous adverse effects of RT and CT, a major limitingfactor is the development of drug resistance by the tumors, andinduction of tumor cell growth arrest and senescence. While senescenttumors do not increase in size per se, they still retain the capacity toproduce and secrete tumor stimulating mitogens and pro-angiogenicfactors that can lead to tumor progression.

The present inventors have previously unexpectedly shown that PABA,which inhibits tyrosinase and melanogenesis, in addition to being atherapeutic for melanotic tumors, can also potentiate treatment ofnon-melanotic carcinomas with RT and CT (see co-pending U.S. ProvisionalApplication Ser. No. 60/538,359 filed Jan. 21, 2004. Accordingly, it washypothesized that benzoic acid derivatives that also inhibit tyrosinasemay also have this activity for both melanotic and non-melanotic cancer.

Cell Cycle Regulation and PXR/SXR Receptors

Induction of cell-cycle arrest and by activation of G1/S and G2/M checkpoints is known to be critical for the capacity of the cell to undergoDNA repair prior to re-entering the cell-cycle. Moreover, studies haveprovided that overriding the cell cycle check point controls in cellswith DNA damage (e.g., induced by RT or CT), can force the cell tore-enter the cell cycle without repairing the damage, leading to mitoticcatastrophe and cell death by apoptosis.

Recent studies have shown that parameters such as the degree ofoxygenation/hypoxia, the expression and function of DNA repair proteins,cell cycle and checkpoint control proteins, and cell adhesion andextracellular matrix proteins all regulate tumor radiosensitivity. Inparticular, in response to DNA-damaging agents such as RT and CT,several kinases are activated (e.g., ATM and ART) which phosphorylatecheck point control proteins such as Chk2, which in turn, phosphorylatesCDC25A, targeting CDC25A for degradation. Degradation of CDC25A leads tocell cycle arrest and DNA repair, while overexpression of CDC25A hasbeen demonstrated to activate Cdk2 kinase, leading to accumulation ofCyclin E/Cck2 complexes and cell cycle progression followed shortly bycell death. Studies have suggested that alteration of cell cycleregulators such as CDC25A, CHK-1 and CHK-2 can sensitize sells to deathby RT and CT, suggesting that agents that inhibit cell cycle arrest orpromote cell cycle transition may be useful as adjuvant therapy for RTor CT.

Induction of cell cycle DNA repair proteins in response to cell cyclearrest is also hypothesized to have a role in radio- andchemosensitivity. Studies have shown that BRCA-2, a DNA repair protein,can regulate transcription of RNA polymerase II, or directly bind toReplication Protein A to regulate DNA repair. Accordingly agents thatfunctionally inactivate or decrease expression of BRCA-2 may enhancecell cycle progression, and hence, enhance sensitivity of tumor cells toCT and RT (Wong et al., Oncogene 2003; 22: 28-33; and Kraakman-van derZwet et al., Mol. Cell Biol. 2002; 22: 669-679).

para-amino-benzoic acid (hereinafter “PABA”), is a water-solublenaturally-occurring compound that is essential for microorganisms andsome animals, but not humans. PABA also has been shown to inhibit cellcycle arrest and DNA repair. Studies in Xenopus embryos have revealed asignaling pathway mediated by endogenous benzoic acid derivatives (i.e.,PABA derivatives such as 3-hydroy ethyl benzoate or 3-HEB) and orphannuclear hormone receptors, benzoate X receptors (BXR). BXRheterodimerizes with the orphan retinoic acid X receptors (RXR) toregulate gene expression. A human and rodent homologue of BXR has beenidentified and is designated Pregnane X receptor or steroid X receptor(PXR/SXR). PXR/SXR is a member of xenobiotic nuclear receptors whichfunction in induction of cytochrome P450 enzymes involved in drugmetabolism and detoxification of xenobiotic compounds, and have limitedexpression restricted to the liver and small intestine. PABA and PABAderivatives have been shown to bind and activate the PXR/SXR receptor(Moore et al., Mol. Endocrinol. 2002; 16: 977-986).

Interestingly, the PXR/SXR receptor has also been found to be abnormallyexpressed in some tumors, such as melanoma (see Moore, supra). Thepresent invention demonstrates that PABA and PABA derivatives which bindto PXR/SXR can surprisingly potentiate tumoricidal effects of CT and RTin tumor cells overexpressing PXR/SXR, likely by inhibiting cell cyclearrest and DNA repair through transcriptional regulation of cell cycleregulatory proteins.

There remains a need in the art for therapies that inhibit cell cyclearrest and DNA repair, and hence, cell senescence specifically in tumorcells, in order to sensitize tumor cells to killing by RT and CT.Accordingly, it is likely that specific PXR/SXR agonists may provide anovel approach to enhance the anti-tumor effects of CT and RT in tumorcells and not normal cells.

SUMMARY OF THE INVENTION

The present invention provides a method of treating cancer, comprisingadministering an effective amount of a benzoic acid derivative.

In on embodiment, the benzoic acid derivative is administered asmonotherapy.

In another embodiment, the benzoic acid derivative is administered incombination with chemotherapy or radiation therapy.

In a preferred embodiment, the benzoic acid derivative is the compounddesignated C-45, depicted in FIG. 1.

In a specific embodiment, the benzoic acid derivative inhibits theenzyme tyrosinase.

The present invention also provides a method for treating cancercomprising cells which express the PXR/SXR xenobiotic receptor, whichmethod comprises administering a compound which is an agonist of thePXR/SXR receptor.

In one embodiment, the PXR/SXR agonist is a benzoic acid derivative.

In another embodiment, the PXR/SXR agonist is a compound that inhibitsexpression or activity of a DNA repair protein and increase expressionor activity of a cell cycle progression protein.

The present invention further provides a method of screening forcompounds which are agonists of the PXR/SXR receptor by i) contactinghost cells containing a reporter gene construct regulated by axenobiotic response element ii) contacting the host cells with a testcompound, and iii) determining the increase in expression of thereporter gene compared to untreated cells harboring the xenobioticresponse element.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 depicts benzoic acid derivatives which can be used totreat cancer according to the method of the present invention.

FIG. 2. FIG. 2 demonstrates the inhibition of tyrosinase over time withPABA and two derivative compounds.

FIG. 3A-C. FIG. 3 shows in vitro effects on the proliferation of B16F10(3A), Lewis Lung carcinoma (3B), and 4T1 (3C) breast carcinoma cellstreated with compound C-45, Taxol®, and a combination of C-45 andTaxol®.

FIG. 4. FIG. 4 shows the in vivo effects of compound C-45, Taxol®, and acombination thereof, on tumor growth in nude mice.

FIG. 5A-B. FIG. 5 demonstrates the effects of C-45, alone, and incombination with radiation—(5A) and chemotherapy (5B), on growth of theB16F10 melanoma tumors in chick embryos.

DETAILED DESCRIPTION

It has now been surprisingly discovered that PABA and specific PABAderivatives which i) inhibit enzymes such as tyrosinase involved inmelanogenesis, or ii) bind to PXR/SXR receptors to regulate genesinvolved in cell cycle progression, can specifically target tumor cells(including non-melanotic tumors) and potentiate tumoricidal activitiesof CT and RT in such tumor cells. Accordingly, identification of leadcompounds with these activities will likely lead to improvedtumor-specific treatment for numerous cancers including melanoma.

The present invention is, in part, based on the findings described inthe Examples. As described in Examples 1, PABA and related derivativeswhich inhibit tyrosinase are shown to enhance tumor cell death mediatedby chemotherapy and ionizing radiation both in vitro and in vivo. Asdescribed in Examples 4-6, benzoic acid derivatives, via agonist effectson nuclear xenobiotic receptor PXR/SXR, can mediate expression ofseveral cell cycle proteins, thereby affecting cell cycle progressionand enhancing apoptotic tumor cell death mediated by chemotherapy andionizing radiation.

Definitions

“para-amino-benzoic-acid” (PABA) is commercially available from, e.g.,Sigma-Aldrich Chemical Co., St. Louis, Mo.

“PABA derivatives” or “benzoic acid derivatives” refer to compoundschemically related to benzoic acid, including alkyl and acyl analogues,chlorobenzoic acids, aminobenzoic acids and nitrobenzoic acids, andsalts and esters thereof.

In a specific embodiment, the compound is the compound designated C-45having the following Formula I:

Other specific derivatives contemplated for use in the present inventionare depicted in FIG. 1.

Additional non-limiting benzoic acid derivatives included but are notlimited to the following: 2-ethoxybenzoic acid, 3,4,5-trimethoxybenzoicacid methyl ester/methyl-3,4,5-trimethoxybenzoate,3,4,5-trimethoxybenzoic acid, 3,5-dinitro-4-hydroxy benzoic acid(4-hydroxy-3,5-dinitrobenzoic acid), 3-acetoxy benzoic acid,4-acetylbenzoic acid, 4-amino-2-chlorobenzoic acid,4-chloro-3-sulfamoylbenzoic acid, 4-phenoxybenzoic acid, isovanillicacid, 2-bromo-5-fluorobenzonitrile, 5-bromo-2-iodobenzonitrile,2,3,4,5,6-pentamethylbenzophenone, 3-nitrobenzophenone,4,4′-dibromobenzophenone, 4-aminobenzophenone, 4-bromo-benzophenone,4-hydroxybenzophenone, 5-amino-2-nitrobenzotrifluoride,2-amino-N-cyclohexyl-N-methylbenzylamine hydrochloride,2-amino-N-cyclohexyl-n-methylbenzylamine, 2-(hydroxymethyl)-benzofuran,2,3-dimethylbenzofuran, 2-acetyl-7-methoxybenzofuran,2-acetylbenzofuran, 2-benzofurancarboxaldehyde, 2-benzofurancarboxylicacid, 2-benzofuranoyl chloride, 6,7-dihydro-4(5H)-benzofuranone,2-methoxy-5-sulfamoylbenzoic acid, 4-(1H-imidazol-1-yl)-benzoic acid,4-(chlorosulfonyl)-benzoic acid, 4-nitrobenzamide, 4-nitrobenzoylchloride, 5-chloro-2-methoxybenzoic acid, 1,4-dihydro-2-methylbenzoicacid, 2,3,5-triiodobenzoic acid, 2,4,5-trimethoxybenzoic acid,2,4-dichloro-5-sulfamoylbenzoic acid, 2,4-dimethoxybenzoic acid, and2,5-dimethoxybenzoic acid.

“Cancer” refers to abnormal, malignant proliferations of cellsoriginating from epithelial cell tissue (carcinomas), blood cells(leukemias, lymphomas, myelomas), connective tissue (sarcomas), or glialor supportive cells (gliomas). In one embodiment, the invention relatesto the treatment of carcinomas and blood cell tumors. In a preferredembodiment, the invention relates to the treatment of lung tumors,breast tumors, ovarian tumors, pancreatic tumors, glioblastoma tumors,and sarcomas.

The term cancer includes but is not limited to the following: acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, carcinoma, aids-related cancers, aids-related lymphoma, analcancer, astrocytoma (cerebellar), bile duct cancer (extrahepatic),bladder cancer, bone cancer (osteosarcoma/malignant fibroushistiocytoma), brain stem glioma, ependymoma, childhood visual pathwayand hypothalamic glioma, breast cancer (including male), bronchialadenomas/carcinoids, carcinoid tumor (gastrointestinal), islet cellcarcinoma, carcinoma of unknown primary origin, central nervous systemlymphoma, cervical cancer, childhood cancers, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic myeloproliferativedisorders, clear cell sarcoma of tendon sheaths, colon cancer,colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,ependymoma, ovarian epithelial cancer, esophageal cancer, Ewing's familyof tumors, extracranial germ cell tumor, extragonadal germ cell tumor,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, germ cell tumor, ovarian germ cell tumor, gestationaltrophoblastic tumor, hairy cell leukemia, head and neck cancer,hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngealcancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oralcavity cancer, liver cancer, lung cancer (non-small cell and smallcell), lymphoma, macroglobulinemia, Waldenström's, malignantmesothelioma, medulloblastoma, melanoma, Merkel cell carcinoma,metastatic squamous neck cancer with occult primary, multiple endocrineneoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosisfungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferativediseases, multiple myeloma, chronic myeloproliferative disorders, nasalcavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,oral cancer, oral cavity and lip cancer, oropharyngeal cancer, ovarianlow malignant potential tumor, pancreatic cancer, paranasal sinus andnasal cavity cancer, parathyroid cancer, penile cancer,pheochromocytoma, pineal and supratentorial primitive neuroectodermaltumors, pituitary tumor, pleuropulmonary blastoma, prostate cancer,rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter,transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, softtissue sarcoma, Sezary syndrome, skin cancer (non-melanotic), smallintestine cancer, supratentorial primitive neuroectodermal tumors,T-cell lymphoma, cutaneous testicular cancer, thymoma, thymic carcinoma,thyroid cancer, urethral-cancer, endometrial uterine cancer, uterinesarcoma, vaginal cancer, vulvar cancer, and Wilm's tumor.

The term also includes childhood cancers of all or any of theabove-identified cancers.

In a specific embodiment, the cancer is melanoma.

The term “radiation therapy” or “radiotherapy” refers to use ofhigh-energy radiation to treat cancer. Radiation therapy includesexternally administered radiation, e.g., external beam radiation therapyfrom a linear accelerator, and brachytherapy, in which the source ofirradiation is placed close to the surface of the body or within a bodycavity. Common radioisotopes used include but are not limited to cesium(¹³⁷Cs), cobalt (⁶⁰Co), iodine (¹³¹I), phosphorus-32 (³²P), gold-198(¹⁹⁸Au), iridium-192 (¹⁹²Ir), yttrium-90 (⁹⁰Y), and palladium-109(¹⁰⁹Pd). Radiation is generally measured in Gray units (Gy), where 1Gy=100 rads.

“Chemotherapy” (CT) refers to treatment with anti-cancer drugs. The termencompasses numerous classes of agents including platinum-based drugs,alkylating agents, anti-metabolites, anti-miotic agents,anti-microtubule agents, plant alkaloids, and anti-tumor antibiotics,kinase inhibitors, proetasome inhibitors, EGFR inhibitors, HERdimerization inhibitors, VEGF inhibitors, and antisense molecules, andincludes antibodies. Such drugs include but are not limited toadriamycin, melphalan, ara-C, BiCNU, busulfan, CCNU, pentostatin, theplatinum-based drugs carboplatin, cisplatin and oxaliplatin,cyclophosphamide, daunorubicin, epirubicin, dacarbazine, 5-fluorouracil(5-FU), fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate,altretamine, mithramycin, mitomycin, bleomycin, chlorambucil,mitoxantrone, nitrogen mustard, mercaptopurine, mitozantrone, paclitaxel(Taxol®), vinblastine, vincristine, vindesine, etoposide, gemcitabine,monoclonal antibodies such as Herceptin®, Rituxan®, Campath®, Zevelin®and Bexxar®, irinotecan, leustatin, vinorelbine, STI-571 (Gleevac®),tamoxifen, docetaxel, topotecan, capecetabine (Xeloda®), raltitrexed,streptozocin, tegafur with uracil, temozolomide, thioguanine, thiotepa,podophyllotoxin, filgristim, profimer sodium, letrozole, amifostine,anastrozole, temozolomide, arsenic trioxide, epithalones A and Btretinioin, interleukins (e.g., 2 and 12) and interferons, e.g., alphaand gamma, bortezomib, huBr-E3, Genasense, Ganite, FIT-3 ligand,MLN491RL, MLN2704, MLN576, and MLN518. Antiangiogenic agents include butare not limited to BMS-275291, Dalteparin (Fragmin®) 2-methoxyestradiol(2-ME), thalodmide, CC-5013 (thalidomide analog), maspin, combretastatinA4 phosphate, LY317615, soy isoflavone (genistein; soy protein isolate),AE-941 (Neovastat™; GW786034), anti-VEGF antibody (Bevacizumab;Avastin™), PTK787/ZK 222584, VEGF-trap, ZD6474, EMD 121974, anti-anb3integrin antibody (Medi-522; Vitaxin™), carboxyamidotriazole (CAI),celecoxib (Celebrex®), halofuginone hydrobromide (Tempostatin™), andRofecoxib (VIOXX®).

The term “chemotherapy” also includes gene therapy with agents such asinterferon and the interleukins, i.e., administration of a vectorencoding genes for the interferons or interleukins. See e.g., Heller etal., Technol Cancer Res Treat. 2002; 1(3):205-9.

The term “PXR/SXR” refers to a mammalian steroid and xenobiotic-sensingnuclear receptor. For example, the nucleotide and amino acid sequencesfor the human PXR/SXR can be found in GenBank Accession No. AY091855, orin Blumberg et al., Genes Dev. 1988; 12(20: 3195-3205.

“Cell cycle regulatory proteins” are those proteins, including enzymes,which are required for progression through the cell cycle, i.e.,mitosis, or arrest of the cell cycle, i.e., senescence. Cell cycleprogression proteins include but are not limited to CDC25A, CDC2, Wee-1,Myt-1, cyclin A, cyclin B, and LATS1, and associated cyclin dependentkinases. Proteins involved in cell cycle arrest, which include DNArepair proteins, include but are not limited to Id-1, Id-2, Id-3, ATM,ATR, p53, BRCA-1, BRCA-2, chk-1, Rad-53, and the cyclin dependent kinaseinhibitors.

As used herein, the terms “treatment” or “treat” mean the lessening orameliorating of at least one abnormal or undesirable conditionassociated with cancer. Treatment may, for example, cause a reduction inthe rate or amount of growth of a tumor. Treatment also includesreducing or ameliorating the undesirable symptoms of cancer. Theforegoing are merely non-limiting examples of the treatment of cancer.In a specific embodiment, the term “treatment” refers to enhancing tumorcell death by RT and/or CT by administering PABA derivatives.

As used herein, a “therapeutically effective amount” of an agent is anamount sufficient to ameliorate at least one symptom associated with apathological, abnormal or otherwise undesirable condition, e.g., cancer,an amount sufficient to prevent or lessen the probability that such acondition will occur or re-occur, or an amount sufficient to delayworsening of such a condition. In one embodiment, the term“therapeutically effective amount” is used to refer to an amount havingantiproliferative effect.

Preferably, the therapeutically effective amount has apoptotic activity,or is capable of inducing cell death, and preferably death of benign ormalignant tumor cells, in particular cancer cells. Efficacy can bemeasured in conventional ways, depending on the condition to be treated.For cancer therapy, efficacy can, for example, be measured by assessingthe time for disease progression, or determining the response rates. Ina preferred embodiment, and effective amount of PABA is an amount thatreduces or inhibits the growth and/or proliferation of tumor cells in anindividual in need of treatment alone, or in combination with RT or CT.

As used herein, the phrase “individual or mammal in need of suchtreatment” refers to a mammal suffering from at least one type ofhyperproliferative disorder or who has been diagnosed with cancer.

The phrase “in combination with” refers to a method of treatment inwhich two or more treatments are administered collectively or accordingto a specific sequence, such that they produce a desirable effect.

The term “potentiate” means to increase the effect of, or actsynergistically with, a drug or a biologic. In one embodiment of thepresent invention, PABA derivatives potentiate the tumorcidal activityor inhibition of tumor growth effected by RT or CT.

As used herein, the term “lead compound” or “candidate compound” is acompound that i) inhibits tyrosinase in melanoma cells, e.g., C-45, orii) binds to and activates the PXR/SXR receptor. In a preferredembodiment, the lead compounds are benzoic acid derivatives, includingbut not limited to those depicted in FIG. 1.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar toxicity (for example, gastric upset,dizziness and the like) when administered to an individual. Preferably,and particularly where a formulation is used in humans, the term“pharmaceutically acceptable” may mean approved by a regulatory agency(for example, the U.S. Food and Drug Agency) or listed in a generallyrecognized pharmacopeia for use in animals (e.g., the U.S.Pharmacopeia).

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils. Water or aqueoussolution saline solutions and aqueous dextrose and glycerol solutionsare preferably employed as carriers, particularly for injectablesolutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin, 18th Edition.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 10- or5-fold, and more preferably within 2-fold of a given value. Numericalquantities given herein are approximate unless stated otherwise, meaningthat the term “about” or “approximately” can be inferred when notexpressly stated.

Molecular Biology

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e., contaminants, including native materials fromwhich the material is obtained. For example, a purified protein ispreferably substantially free of other proteins or nucleic acids withwhich it is associated in a cell; a purified nucleic acid molecule ispreferably substantially free of proteins or other unrelated nucleicacid molecules with which it can be found within a cell. As used herein,the term “substantially free” is used operationally, in the context ofanalytical testing of the material. Preferably, purified materialsubstantially free of contaminants is at least 95% pure; morepreferably, at least 97% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art. In a specific embodiment, purified means that thelevel of contaminants is below a level acceptable to regulatoryauthorities for administration to a human or non-human animal.

A “gene” is a sequence of nucleotides which code for a functional “geneproduct”. Generally, a gene product is a functional protein. However, agene product can also be another type of molecule in a cell, such as anRNA (e.g., a tRNA or a rRNA). For the purposes of the present invention,a gene product also refers to an mRNA sequence which may be found in acell.

The term “express” and “expression” means allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing RNA (such as rRNA or mRNA) or a protein by activating thecellular functions involved in transcription and translation of acorresponding gene or DNA sequence. A DNA sequence is expressed by acell to form an “expression product” such as an RNA (e.g., a mRNA or arRNA) or a protein. The expression product itself, e.g., the resultingRNA or protein, may also said to be “expressed” by the cell.

“Expression level” correspond to levels of a detectable cellular producte.g., mRNA or a corresponding gene product, or an activity of such agene product. For example, according to the screening method of presentinvention, changes in the expression level of a reporter geneoperatively associated with the PXR/SXR response element can be used todetermine whether a compound binds to and activates the PXR/SXRreceptor.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequenceinto a host cell so that the host cell will express the introduced geneor sequence to produce a desired substance, in this invention typicallyan RNA coded by the introduced gene or sequence, but also a protein oran enzyme coded by the introduced gene or sequence. The introduced geneor sequence may also be called a “cloned” or “foreign” gene or sequence,may include regulatory or control sequences (e.g., start, stop,promoter, signal, secretion or other sequences used by a cell's geneticmachinery). The gene or sequence may include nonfunctional sequences orsequences with no known function. A host cell that receives andexpresses introduced DNA or RNA has been “transformed” and is a“transformant” or a “clone”. The DNA or RNA introduced to a host cellcan come from any source, including cells of the same genus or speciesas the host cell or cells of a different genus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g., a foreign gene) can beintroduced into a host cell so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence.

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g., for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, insect host cells such as Sf9, Hi5 or S2 cells and Baculovirusvectors, and expression systems, and mammalian host cells, includingtumor cells and cell lines, and vectors.

By “vector” is meant any genetic element, such as a plasmid, phage,transposon, cosmid, chromosome, virus, virion, etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

Screening

The ability to screen compounds for the capability to inhibittyrosinase, or to bind and activate the PXR/SXR receptors, will permitthe identification and selection of lead compounds for the improvedtreatment of melanoma and other cancers, or for the treatment of tumorswhich express PXR/SXR receptors when combined with standard treatment,e.g., CT or RT. For the latter, compounds that are preferred are thosethat differentially up-regulate cell cycle progression proteins such asCDC25A, and down-regulate DNA repair proteins such as BRCA-2.

Screening can be achieved by any method known in the art, including butnot limited to the methods described below in the Examples, by usingcell-free and cell-based, and high-throughput methods described furtherbelow.

In vitro Screening Methods

Various assays can be designed to screen for PABA-like inhibitors oftyrosinase, or agonists of PXR/SXR. Although in vitro methods arepreferred for any initial screening of large number of potential drugcandidates or agents, the in vivo methods described below may also beused for screening.

Tyrosinase inhibitors. The tyrosinase inhibitors may be both direct andindirect inhibitors. Preferred, although non-limiting, examples ofindirect inhibitors include anti-sense nucleic acids complementary togenomic DNA or mRNA encoding tyrosinase, thus preventing translation ofthe coding nucleic acid sequences into the target protein. Methods todesign and screen for antisense nucleic acids are well-known in the art.Thus, anti-sense sequences may be used to modulate the activity of thedrug target or to achieve regulation of gene function. Sense oranti-sense oligomers, or larger fragments, can be designed from variouslocations along the coding or regulatory regions of sequences encoding adrug target of the invention.

Alternative indirect inhibitors include compounds, such as theexemplified benzoic acid derivative C-45, that reduce transcription ofthe gene encoding the target protein, i.e., tyrosinase.

Direct inhibitors of tyrosinase can be identified by evaluating theinhibitory effect of a drug candidate or test agent on the biologicalactivity of the selected target protein (“drug target”) in comparison toa control or reference. The control or reference may be a predeterminedreference value, or may be evaluated experimentally. For example, thecontrol or reference value can be a measure of the biological activityof the target protein in the absence of the test agent, or thebiological activity of a reference protein in the presence of testagent, or any other suitable control or reference.

Drugs or agents that inhibit the activity of a target protein, e.g.,tyrosinase, can be identified based on their ability to associate withthe drug target protein. Association with a drug target can be tested byreacting a drug target protein or fragment with a test substance whichhas the potential to associate with the drug target under appropriateconditions, and removing and/or detecting the associated drugtarget/test substance complex. Binding may be detected by indirect ordirect functional measures such as alteration of migration pattern inprotein gel electrophoresis, immunoprecipitation, or the BiomolecularInteraction Assay (BIAcore; Pharmacia). A drug candidate that associateswith a drug target protein of the invention is preferably an antagonistor inhibitor of the biological activity of a drug target, as shown by anactivity assay.

Activity assays are generally designed to measure the activity of atarget protein in the presence or absence of a test agent. Manydifferent activity assays may be designed based on variousart-recognized methods for studying the activity of tyrosinase. Forexample, as described in Example 1, inhibitors of tyrosinase activitycan be identified by measuring the ability of tyrosinase to promote theconversion of a L-DOPA into a DOPAochrome over a suitable period oftime, as detected by measuring absorbance. Optionally, in cases wherethe substrate is detectable by fluorescence or by coloring, the amountof intact substrate remaining can be measured after incubation forsuitable time period.

PXR/SXR agonists. To assess PXR/SXR binding and activation by testcompounds and other benzoic acid derivatives, standard cell-basedreporter assays can be employed. This technique involves transfectingtarget cells with a reporter construct carrying the xenobiotic responseelements (e.g., DR3 motif 5′ AGTTCA 3′) upstream of e.g., a luciferasereporter gene. Following treatment of cells with e.g., PABA or any othertest compound, activation of the PXR/SXR-receptors will be quantified bymeasuring the increase in luciferase activity as a result of activationof the receptors and their binding to the xenobiotic response elements(Kliewer et al., Cell 1998; 92: 73-82; and Takeshita et al., J. Biol.Chem. 2002; 277: 32453-8).

In addition, screening for compounds affecting PXR/SXR binding oractivity in the presence of test substances, additional reporter geneassays which be used include a green fluorescent protein expressionsystem, and modifications thereof (U.S. Pat. No. 5,625,048 and PCTPublication No. WO 98/06737; PCT Publication No. WO 96/23898). Otherreporter genes include β-galactosidase (β-gal or lac-Z), chloramphenicoltransferase (CAT), horseradish peroxidase, and alkaline phosphatase.

In addition, levels of CDC25A and BRCA-2 and other cell cycle-relatedproteins, such as Id-1, Id-2 and Id-3, in response to drug treatment canbe determined using specific antibodies, e.g., by standardimmunoblotting or other quantification assays well known in the art.

Nucleic acid expression, such as mRNA expression, can be determined bystandard RT-PCR and/or Northern hybridization techniques.

In addition to screening, the role of the PXR/SXR receptor in PABAinduced radio- and chemosensitization can be further elucidated bysilencing the PXR/SXR receptor, e.g., using siRNA technology, andassessing the effect of PABA and related derivative compounds on cellcycle-associated proteins such as CDC25A, Id-1, Id-2, Id-3 and BRCA-2.

Compound Libraries

In addition to the PABA-derivatives described herein as PXR/SXRagonists, synthetic libraries provide a source of potential agonistsaccording to the present invention libraries (Needels et al., Proc.Natl. Acad. Sci. USA 1993, 90:10700-4; Ohlmeyer et al., Proc. Natl.Acad. Sci. USA 1993, 90:10922-10926; Lam et al., PCT Publication No. WO92/00252; Kocis et al., PCT Publication No. WO 9428028). Syntheticcompound libraries are commercially available from Maybridge ChemicalCo. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), BrandonAssociates (Merrimack, N.H.), and Microsource (New Milford, Conn.). Arare chemical library is available from Aldrich (Milwaukee, Wis.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available, e.g., from PanLaboratories (Bothell, Wash.) or MycoSearch (NC), or are readilyproducible. Additionally, natural and synthetically produced librariesand compounds are readily modified through conventional chemical,physical, and biochemical means (Blondelle et al., TIBTech 1996, 14:60).

Another approach uses recombinant bacteriophages to produce largelibraries. Using the “phage method”, very large libraries can beconstructed (106-108 chemical entities) (Scott and Smith, Science 1990,249:386-390; Cwirla, et al., Proc. Natl. Acad. Sci. USA 1990,87:6378-6382; Devlin et al., Science 1990, 49:404-406). A alternativeapproach uses primarily chemical methods, of which the Geysen method(Geysen et al., Molecular Immunology 1986, 23:709-715; Geysen et al. J.Immunologic Methods 1987, 102:259-274); and the method of Fodor et al.(Science 1991, 251:767-773) are examples.

Classes of compounds that may be identified by such screening assaysinclude, but are not limited to, small molecules (e.g., organic orinorganic molecules which are less than about 2 kd in molecular weight,are more preferably less than about 1 kD in molecular weight, and/or areable to cross the blood-brain barrier or gain entry into an appropriatecell, as well as macromolecules (e.g., molecules greater than about 2 kDin molecular weight).

The compounds used in such screening assays are also preferablyessential pure and free of contaminants that may, themselves, alter orinfluence gene expression. Compound purity may be assessed by any numberof means that are routine in the art, such as LC-MS and NMRspectroscopy. Libraries of test compounds are also preferably biased byusing computational selection methods that are routine in the art. Toolsfor such computational selection, such as Pipeline Pilot™ (ScitegicInc., San Diego, Calif.) are commercially available. The compounds maybe assessed using rules such as the “Lipinski criteria” (see, Lipinskiet al., Adv. Drug Deliv. Rev. 2001, 46:3-26) and/or an other criteria ormetrics commonly used in the arts.

High-Throughput Screening

The in vitro assay systems described here may be used in ahigh-throughput primary screen for compounds. For example, drugcandidates according to the invention may advantageously be identifiedby screening in high-throughput assays, including without limitationcell-based or cell-free assays. It will be appreciated by those skilledin the art that different types of assays can be used to detectdifferent types of drugs or agents. Several methods of automated assayshave been developed in recent years so as to permit screening of tens ofthousands of compounds in a short period of time (see, e.g., U.S. Pat.Nos. 6,303,322, 5,585,277, 5,679,582, and 6,020,141). Suchhigh-throughput screening methods are particularly preferred.Identifying agents is greatly facilitated by use of high-throughputscreening assays to test for agents together with large amounts of drugcandidates, provided as described herein.

In vivo Screening Methods

The in vivo chick embryo and mouse xenograft models of the invention canadvantageously be used for testing the efficacy of a drug identified asa candidate drug in an in vitro screen, and for optimizing dosages andadministration schedules of the drug candidate to enhanceanti-proliferative effects of CT and RT. In one embodiment of theinvention, drugs that inhibit the activity of tyrosinase or agonizeactivity of the PXR/SXR receptor, and are therefore candidates forpotentiating the tumoricidal effects of CT and RT, are evaluated asdescribed in Examples 1-6 below.

Formulations and Administration Formulations

For use in the present invention, PABA may be formulated into apharmaceutical composition. The pharmaceutical composition may includeadditives, such as a pharmaceutically acceptable carrier or diluent, aflavorant, a sweetener, a preservative, a dye, a binder, a suspendingagent, a dispersing agent, a colorant, a disintegrant, an excipient, afilm forming agent, a lubricant, a plasticizer, an edible oil or anycombination of two or more of the foregoing.

Suitable pharmaceutically acceptable carriers or diluents include, butare not limited to, ethanol, water, glycerol, propylene glycol, aloevera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, PPG2myristyl propionate, magnesium carbonate, potassium phosphate, vegetableoil, animal oil, and solketal. Preferred carriers are vegetable andmineral oils.

Suitable binders include, but are not limited to, starch, gelatin,natural sugars, such as glucose, sucrose and lactose; corn sweeteners,natural and synthetic gums, such as acacia, tragacanth, vegetable gum,and sodium alginate, carboxymethylcellulose,hydroxypropylmethylcellulose, polyethylene glycol, povidone, waxes; andthe like.

Suitable disintegrants include, but are not limited to, starch, e.g.,corn starch, methyl cellulose, agar, bentonite, xanthan gum, sodiumstarch glycolate, crosspovidone and the like.

Suitable lubricants include, but are not limited to, sodium oleate,sodium stearate, sodium stearyl fumarate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like.

A suitable suspending agent is, but is not limited to, bentonite,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, agar-agarand tragacanth, or mixtures of two or more of these substances, and thelike.

Suitable dispersing and suspending agents include, but are not limitedto, synthetic and natural gums, such as vegetable gum, tragacanth,acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinyl-pyrrolidone and gelatin.

Suitable film forming agents include, but are not limited to,hydroxypropylmethylcellulose, ethylcellulose and polymethacrylates.

Suitable plasticizers include, but are not limited to, polyethyleneglycols of different molecular weights (e.g., 200-8000 Da) and propyleneglycol.

Suitable colorants include, but are not limited to, ferric oxide(s),titanium dioxide and natural and synthetic-lakes.

Suitable edible oils include, but are not limited to, cottonseed oil,sesame oil, coconut oil and peanut oil.

Examples of additional additives include, but are not limited to,sorbitol, talc, stearic acid, dicalcium phosphate and polydextrose.

Dosages and Dosage Forms

The pharmaceutical composition or unit dosage form of the presentinvention, i.e., a benzoic acid derivative such as C-45, may beadministered according to a dosage and administration regimen defined byroutine testing in order to obtain optimal activity while minimizingtoxicity or side-effects for a particular patient. Typically, dosageswill determined by those skilled in the art on a case-by-case basis,depending upon the tumor type, stage, location, and prognosis of theindividual, and other factors such as weight, sex and age of theindividual, the particular dosage form employed, and the route ofadministration utilized. Pharmacokinetics and pharmacodynamics such ashalf-life (t_(1/2)), peak plasma concentration (c_(max)), time to peakplasma concentration (t_(max)), and exposure as measured by area underthe curve (AUC) can be obtained using ordinary methods known in the art.

Data obtained from cell culture assay or animal studies may be used toformulate a therapeutic dosage range for use in humans and non-humananimals. The dosage of compounds used in therapeutic methods of thepresent invention preferably lie within a range of circulatingconcentrations that includes the ED₅₀ concentration (effective for 50%of the tested population) but with little or no toxicity.

A therapeutically effective dose may be initially estimated from cellculture assays and formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀. The IC₅₀ concentration of acompound is the concentration that achieves a half-maximal inhibition ofsymptoms (e.g., as determined from the cell culture assays). Appropriatedosages for use in a particular individual, for example in humanpatients, may then be more accurately determined using such information.

In one embodiment, the compound may be administered, alone or incombination with CT or RT in the range of about 10 g/day, preferably ina range from about 10 mg/day to about 6 g/day, more preferably in arange from about 250 mg/day to about 5 g/day.

For combination therapy with radiation, the radiation is typicallyadministered in doses of 1 cGy to 100 Gy. More preferably, radiation isadministered in doses of 2 cGy to 20 Gy. Factors such as dose ratedelivered, tumor size, and radiosensitivity play a major role indetermining therapeutic response, while target-to-nontarget ratios and,particularly, circulating radioactivity to the bone marrow determine themajor dose-limiting toxicities.

Dosages of chemotherapy are not only drug-type specific, but also dependto a large extent on the individual patient, the tumor-type, and thestage of the disease, and accordingly, are determined by one of ordinaryskill in the art. By way of example, standard doses of paclitaxel(Taxol®) in combination with carboplatin for ovarian and lung cancer are175 mg/m² of the former and AUC 5 mg/ml*min of the latter.

Unit dosage forms. The above compositions of the benzoic acidderivatives may be formulated as unit dosage forms such as tablets,pills, capsules, caplets, boluses, powders, granules, sterile parenteralsolutions, sterile parenteral suspensions, sterile parenteral emulsions,elixirs, tinctures, metered aerosol or liquid sprays, drops, ampoules,autoinjector devices or suppositories. Unit dosage forms may be used fororal, parenteral, intranasal, sublingual or rectal administration, orfor administration by inhalation or insufflation, transdermal patches,and a lyophilized composition. In general, any delivery of activeingredients that results in systemic availability of them can be used.

Preferably the unit dosage form of the benzoic acid derivative is anoral dosage form, most preferably a solid oral dosage form, thereforethe preferred dosage forms are tablets, pills, caplets and capsules.PABA-derivative-containing solutions and suspensions for oraladministration are also preferred. However, the compound can also beformulated for parenteral administration. Parenteral preparations (e.g.,injectable preparations in saline and preparations for powder jetsystems) are preferred for CT and RIT.

Solid unit dosage forms may be prepared by mixing an active agent of thepresent invention with a pharmaceutically acceptable carrier and anyother desired additives as described above. The mixture is typicallymixed until a homogeneous mixture of the active agents of the presentinvention and the carrier and any other desired additives is formed,i.e., until the active agent is dispersed evenly throughout thecomposition. In this case, the compositions can be formed as dry ormoist granules.

Dosage forms with predetermined amounts of the benzoic acid derivativesmay be formulated starting with compositions with known quantities ofthe compounds using methods well known in the art. In a preferredembodiment a dosage form is obtained by mixing compositions comprisingknown quantities of the derivatives.

Dosage forms can be formulated as, for example, “immediate release”dosage forms. “Immediate release” dosage forms are typically formulatedas tablets that release at least 70%-90% of the active ingredient within30-60 min when tested in a drug dissolution test, e.g., U.S.Pharmacopeia standard <711>. In a preferred embodiment, immediate dosageforms release 75% of active ingredients in 45 min.

Dosage forms can also be formulated as, for example, “controlledrelease” dosage forms. “Controlled,” “sustained,” “extended” or “timerelease” dosage forms are equivalent terms that describe the type ofactive agent delivery that occurs when the active agent is released froma delivery vehicle at an ascertainable and modifiable rate over a periodof time, which is generally on the order of minutes, hours or days,typically ranging from about sixty minutes to about 3 days, rather thanbeing dispersed immediately upon entry into the digestive tract or uponcontact with gastric fluid. A controlled release rate can vary as afunction of a multiplicity of factors. Factors influencing the rate ofdelivery in controlled release include the particle size, composition,porosity, charge structure, and degree of hydration of the deliveryvehicle and the active ingredient(s), the acidity of the environment(either internal or external to the delivery vehicle), and thesolubility of the active agent in the physiological environment, i.e.,the particular location along the digestive tract. Typical parametersfor dissolution test of controlled release forms are found in U.S.Pharmacopeia standard chapter <724>.

Dosage forms can also be formulated to deliver active agent inmultiphasic stages whereby a first fraction of an active ingredient isreleased at a first rate and at least a second fraction of activeingredient is released at a second rate. In a preferred embodiment, adosage form can be formulated to deliver active agent in a biphasicmanner, comprising a first “immediate release phase”, wherein a fractionof active ingredient is delivered at a rate set forth above forimmediate release dosage forms, and a second “controlled release phase,”wherein the remainder of the active ingredient is released in acontrolled release manner, as set forth above for controlled releasedosage forms.

Tablets or pills can be coated or otherwise compounded to form a unitdosage form which has delayed and/or prolonged action, such as timerelease and controlled release unit dosage forms. For example, thetablet or pill can comprise an inner dosage and an outer dosagecomponent, the latter being in the form of a layer or envelope over theformer. The two components can be separated by an enteric layer whichserves to resist disintegration in the stomach and permits the innercomponent to pass intact into the duodenum or to be delayed in release.

Biodegradable polymers for controlling the release of the active agents,include, but are not limited to, polylactic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathicblock copolymers of hydrogels.

For liquid dosage forms, the active substances or their physiologicallyacceptable salts are brought into solution, suspension or emulsion,optionally with the usually employed substances such as solubilizers,emulsifiers or other auxiliaries. Solvents for the active combinationsand the corresponding physiologically acceptable salts, can includewater, physiological salt solutions or alcohols, e.g. ethanol,propane-diol or glycerol. Additionally, sugar solutions such as glucoseor mannitol solutions may be used. A mixture of the various solventsmentioned may further be used in the present invention.

A transdermal dosage form also is contemplated by the present invention.Transdermal forms may be a diffusion-driven transdermal system(transdermal patch) using either a fluid reservoir or a drug-in-adhesivematrix system. Other transdermal dosage forms include, but are notlimited to, topical gels, lotions, ointments, transmucosal systems anddevices, and iontohoretic (electrical diffusion) delivery system.Transdermal dosage forms may be used for timed release and controlledrelease of the active agents of the present invention.

Pharmaceutical compositions and unit dosage forms of the presentinvention for administration parenterally, and in particular byinjection, typically include a pharmaceutically acceptable carrier, asdescribed above. A preferred liquid carrier is vegetable oil. Injectionmay be, for example, intravenous, intrathecal, intramuscular,intratracheal, or subcutaneous.

The active agent also can be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

The benzoic acid derivatives of the present invention also may becoupled with soluble polymers as targetable drug carriers. Such polymersinclude, but are not limited to, polyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amidephenol,polyhydroxy-ethylaspartamidephenol, and polyethyl-eneoxideopolylysinesubstituted with palmitoyl residues.

Administration

The pharmaceutical composition or unit dosage forms of the presentinvention may be administered by a variety of routes such asintravenous, intratracheal, subcutaneous, oral, intratumoral, mucosalparenteral, buccal, sublingual, rectal, ophthalmic, pulmonary,transmucosal, transdermal, and intramuscular. Unit dosage forms also canbe administered in intranasal form via topical use of suitableintranasal vehicles, or via transdermal routes, using those forms oftransdermal skin patches known to those of ordinary skill in the art.Oral administration of the derivatives is preferred. Also preferred isadministration by local intratumoral injection.

The pharmaceutical composition or unit dosage forms of the presentinvention may be administered to a mammal, preferably a human being, inneed of cancer treatment.

The pharmaceutical composition or unit dosage form may be administeredin a single daily dose, or the total daily dosage may be administered individed doses. In addition, co-administration or sequentialadministration of other active agents may be desirable. The derivativesand mixtures thereof of the invention may be combined with any knowndrug therapy, preferably RT and/or CT, for the treatment of cancer.

The pharmaceutical composition or unit dosage form may be administeredin a single daily dose, or the total daily dosage may be administered individed doses. The combination of the benzoic acid derivative and RT orCT may be co-administered simultaneously or sequentially administered.The compounds preferably will be provided as separate dosage forms.

The exact dosage and administration regimen utilizing the combinationtherapy of the present invention is selected in accordance with avariety of factors including type, species, age, weight, sex and medicalcondition of the patient; the route of administration; the renal andhepatic function of the patient; the treatment history of the patient;and the responsiveness of the patient. Optimal precision in achievingconcentrations of compounds within the range that yields efficacywithout toxicity requires a regimen based on the kinetics of the drug'savailability to target sites. This involves a consideration of theabsorption, distribution, metabolism, excretion of a drug, andresponsiveness of the patient to the dosage regimen. However, such finetuning of the therapeutic regimen is routine in light of the guidelinesgiven herein.

EXAMPLES

The present invention is further described by means of the examples,presented below. The use of such examples is illustrative only and in noway limits the scope and meaning of the invention or of any exemplifiedterm. Likewise, the invention is not limited to any particular preferredembodiments described herein. Indeed, many modifications and variationsof the invention will be apparent to those skilled in the art uponreading this specification and can be made without departing from itsspirit and scope. The invention is therefore to be limited only by theterms of the appended claims along with the full scope of equivalents towhich the claims are entitled.

Example 1 PABA and Derivatives Inhibit Tyrosinase Activity in vitroMethods

Activity of purified tyrosinase was measured by monitoring the formationof DOPAchrome. Briefly, a fixed concentration of L-DOPA (8.0 mM) wasincubated with 800 U/ml tyrosinase (from mushrooms-Sigma, St. Louis,Mo.) in the absence or presence of increasing concentrations of PABA,compound 44 (C-44) and compound 45 (C-45) (see FIG. 1 for structures)for indicated time points (see FIG. 2 for concentrations and timepoints). At each time point, 100 μl of the reaction mixture wasmeasured. Data points represent the mean optical density at 475 nm±SDfrom triplicate wells.

Results

As shown in FIG. 2, PABA, C-44, and C-45 all inhibited tyrosinaseactivity at 8.0 mM up to 10 minutes, the last time point measured. Bycontrast, untreated samples demonstrated linearly increasing tyrosinaseactivity up to 10 minutes. This approach can be applied to evaluateother benzoic acid derivatives or other compounds for inhibitoryactivity on tyrosinase.

Example 2 Effects of PABA Derivative C-45 on Carcinoma Cells Treatedwith Chemotherapy Methods

Tumor cell proliferation was assessed by monitoring mitochondrialdehydrogenase activity as detected by tetrazolium salt cleavage using acommercially available kit (WST-1; Roche, Indianapolis, Ind.). Briefly,B16F10 melanoma cells, Lewis Lung carcinoma cells, and 4T1 breastcarcinoma cells (1×10⁶) were resuspended in PABA-free growth mediumsupplemented with 2.0% fetal bovine serum and 50 μg/ml of test compound(as shown in FIG. 1) Following incubation for 3 days, the cells wereeither treated or not treated with 0.01 μM Taxol® and incubated for anadditional 48 hours. Proliferation was measured; data points representthe mean optical density at 490 nm±SD from triplicate wells.

In addition, tumor growth in vivo of xenografts of B16F10 melanoma cellsin nude mice was determined. Briefly, subconfluent cultures of B16F10cells were harvested, washed, and resuspended in sterile PBS. Femalenude mice were injected subcutaneously with 1×10⁶ cells per mouse andtumors were allowed to grow for 3 days. After 3 days, mice were eitheruntreated or treated daily for 15 days by intraperitoneal injection ofTaxol® (0.5 μM), C-45 (100 μg), or a combination of Taxol® and C-45.Tumor growth was determined weekly by caliper measurements. After 15days, the mice were sacrificed and tumor volume was determined bystandard methods using the formula V=L×W/2, where V=volume, L=length,and W=width.

Results

FIG. 3A-C demonstrate that both Taxol® and compound C-45 individuallyinhibited in vitro proliferation of B16F10, Lewis Lung carcinoma, and4T1 breast carcinoma cells compared to untreated control, and theinhibition was even more pronounced with a combination of C-45 andTaxol®.

As shown in FIG. 4, neither Taxol® nor C-45 inhibited in vivo tumorgrowth compared to untreated controls. By contrast, a combination ofTaxol® and C-45 inhibited in vivo growth by more than about 33%.

Example 3 The Effect of C-45 on Radio- and Chemosensitivity in vivoMethods

The chick embryo tumor growth model was used to determine whether C-45enhances the ability of Taxol® or ionizing radiation to inhibit tumorgrowth. Briefly, B16F10 melanoma cells were cultured in growth medium inthe presence or absence of 50 μg/ml C-45 for at least 7 days. Followingharvesting and washing, about 2×10⁵ tumor cells were implanted onto thechorioallontoic membranes of 10 day-old chick embryos and incubated forat least 24 hours. Following incubation, the embryos were treated witheither a single fractionated dose of radiation (5.0 Gy) or Taxol® (0.01μM) and incubated for an additional 7 days. The embryos were sacrificedand tumors were resected and weighed (5-10 embryos per condition).

Results

FIG. 5A-B demonstrates the effect of C-45 on radiation- andchemotherapy-treated tumor growth in chick embryos. As shown in FIG. 5A,while treatment with radiation and C-45 alone reduced tumor weightsignificantly, the combination of radiation and C-45 was even moreeffective at inhibiting tumor growth in vivo. Similarly, while Taxol®and C-45 individually inhibited the growth of the B16F10 tumorssignificantly, the combination was far more effective (FIG. 5B).

Example 4 Screening for Compounds that Bind to and Activate the PXR/SXRReceptor Rationale

It has been previously demonstrated that PABA and other benzoic acidderivatives bind to the PXR/SXR receptor (Moore et al., Mol. Endocrinol.2002; 16: 977-86). The present inventors have shown that when treatedwith PABA, G361 human melanoma cells exhibit decreased proteinexpression of DNA repair protein BRCA-2 and increase expression ofcell-cycle progression checkpoint protein CDC25A (data not shown). G361melanoma cells also express the PXR/SXR receptor whereas non-transformedmelanocytes do not (data not shown). Accordingly, it is expected thatPABA-like benzoic acid derivatives may have similar agonist activity atthis receptor may be useful in the treatment of PXR/SXR over-expressingtumors.

Methods

To assess PXR/SXR binding and activation by test compounds and otherbenzoic acid derivatives, standard cell-based reporter assays can beemployed. For example, target cells can be transfected with a reporterconstruct carrying the xenobiotic response elements (e.g., DR3 motif 5′AGTTCA 3′) upstream of e.g., a luciferase reporter gene. Followingtreatment of cells with e.g., PABA or any other test compound,activation of the PXR/SXR receptors will be quantified by measuring theincrease in luciferase activity as a result of activation of thereceptors and their binding to the xenobiotic response elements (Klieweret al., Cell 1998; 92: 73-82; and Takeshita et al., J. Biol. Chem. 2002;277: 32453-8).

Example 5 Determining Expression of PXR/SXR and Cell Cycle RegulatoryProteins in Tumor Cells in Response to Drug Treatment Methods

To determine whether tumor cells abnormally express and signal throughthe PXR/SXR receptor compared to their non-transformed counterparts, andthus, candidates for treatment with PXR/SXR agonists, immortalized,non-transformed cells and sub-confluent transformed, tumorigenic cellswill be cultured in the presence or absence of various concentrations ofa known PXR/SXR activator, e.g., benzoic acid derivatives, and anegative control, e.g., streptomycin, for a pre-determined time e.g., 5days. Cell lyses will be achieved as described above. Following lyses,about 15 μg/lane of protein will be loaded and separated by 10%SDS-PAGE, followed by transfer to nitrocellulose membranes. Membraneswill then incubated in the presence of e.g., monoclonal antibody 39048,specific for the PXR/SXR nuclear receptor, followed by washing andlabeling with a peroxidase-labeled secondary antibody.

In addition, RT-PCR and/or Northern hybridization can be used todetermine nucleic acid expression, e.g., mRNA expression of the PXR/SXRreceptor. For example, for RT-PCR primers were designed from the humanPXR/SXR cDNA sequence (GenBank Accession No. AY091855) are as follows:5′ Forward AGCAATTCGCCATTACT (SEQ ID NO: 2) 3′ ReverseGCTACCTGTGATACCGAAC (SEQ ID NO: 3)Using a standard amplification protocol consisting of e.g., 40 cycles of94° denaturation for 30 sec, 55° annealing for 30 sec and 72°amplification for 1 min will yield a product of about 203 bp. Detectioncan be achieved by, e.g., electrophoresis on a 2% agarose gel stainedwith ethidium bromide.

Similarly, to evaluate expression of cell cycle proteins that may beregulated by PXR/SXR activation in response to drug treatment, membranesas prepared above will be incubated with commercially availablemonoclonal antibodies such as PC410 (Chemicon, Temecula, Calif.),directed against β-integrin, as a positive control, monoclonal antibody3303, specific for BRCA-2 (QED Biosciences, San Diego, Calif.), and withmonoclonal antibody AB-3, directed against CDC25A (NeoMakers Inc).Additional antibodies may include anti-Id-1 monoclonal antibody B30-1,anti-Id-2 monoclonal antibody B31-1 (BD PharMingen, San Jose, Calif.)and Id-3 monoclonal antibody 56209 (QED Biosciences, San Diego, Calif.).Peroxidase-labeled goat anti-mouse secondary antibodies will then beincubated after washing. Additional controls consisted of membranesprobed with only either the primary or secondary antibodies.

Detection will be performed using laser scanning densitometry using aNuclear Vision 760 system to compare relative expression levels of theproteins.

Results

As stated above, it has been previously demonstrated that G361melanomas, but not immortalized but untransformed melanocytes, expresshigh levels of PXR/SXR (data not shown), and increased levels of CDC25Aand decreased levels of DNA repair protein BRCA-2 when treated withPXR/SXR agonist PABA. This suggests that therapeutic agents like PABA orrelated derivatives which are agonists for this receptor will be usefulin the treatment of PXR/SXR over-expressing tumors.

This finding led to the hypothesis that that PABA derivatives may alsoenhance the effects of chemotherapy and ionizing radiation in a mannersimilar to previously shown by PABA, by preventing tumor cellssenescence upon treatment. A compound that simultaneously inhibits cellcycle repair proteins and increasing the concentration of proteins thatinduce cell cycle progression will be a preferred drug candidate.Accordingly, it is expected that similar effects on DNA repair and cellcycle regulatory protein expression will be observed with other PXR/SXRagonists depicted in FIG. 1 or identified by the screening methods ofthe present invention.

Example 6 Effects of Candidate Compounds on the Cell Cycle andAnti-Proliferative Effects of Ionizing Radiation or Chemotherapy invitro Methods

To test the hypothesis stated above for benzoic acid derivatives thatbind to the PXR/SXR receptor, cell cycle analysis can be performed asfollows using Fluorescent Activated Cell Sorting (FACS) in cells treatedwith drug candidates and CT or RT. Immortalized cells andPXR/SXR-expressing transformed tumorigenic cells will be cultured in thepresence or absence of various concentrations of a test compound, andpositive and negative control compounds (e.g., PABA and streptomycin)for a designated time period. Next, cells will be synchronized by serumstarvation for at least 24 hours, then cultured in serum-containingmedium and treated with a test compound and control compounds, with orwithout various single fraction doses of radiation (e.g., 1, 2, 5, 10and 20 Gy) or chemotherapy. At various time points (e.g., 1, 3, 6, 12and 24 h) following serum stimulation, cells will be removed and washedwith PBS and fixed for 1 hr with 70% ethanol at 4° C. Cells will then bewashed several times with PBS and resuspended in RNAse staining buffer,commercially available from BD PharMingen, for 30 minutes on ice. Cellcycle analysis will be determined using a FACs Scan flow cytometer(Becton-Dickenson, San Jose, Calif.), and data collected using e.g., aHewlett Packard HP9000 equipped with FAC-Scan software and analyzedusing PC-Lysis software (also from Becton-Dickenson). At least 10,000events will be collected with identical gating parameters on single cellpopulations and percentages of cells within G0/G1, S, G2 and Mdetermined along with the apoptotic population.

In addition to cell cycle analysis, cell proliferation can be measuredusing methods such as e.g., tetrazolium salt cleavage or incorporationof tritiated thymidine. For example, cells can be treated with test andcontrol compounds, plated and incubated for 2 hours to permit celladhesion. Plates will then treated or not with various concentrations ofionizing radiation, e.g., 1, 2 10 and 20 Gy, or chemotherapy e.g., 0-0.2μM Taxol®. Proliferation of the cells will then measured using e.g., theWST-1 kit (tetrazolium salt cleavage). Data points representing the meanoptical density at 490 nm from triplicate wells ±SD will be obtained.

Results

It is expected that treatment of non-transformed cells, or cells notexpressing the PXR/SXR receptor, will not have altered proliferationwhen contacted with test compounds, radiation or chemotherapy, or acombination of the test compound with either agent. To the contrary,tumor cells which express PXR/SXR will respond to agonist compounds, asdemonstrated by increased cell death, which will be more pronounced incombination with radiation or chemotherapy. Such compounds will bedesignated candidate or lead compounds.

CONCLUSION

The results presented herein are the first demonstration that benzoicacid derivatives can potentiate radio- and chemosensitivity of tumorcells which overexpress the PXR/SXR nuclear receptor. Given thepotential of PABA to induce radiosensitivity via a PXR/SXR dependentmechanism, taken together with the relatively limited expression ofPXR/SXR receptor in normal cell types, it is likely that specificagonists will provide a novel, effective approach to enhance theanti-tumor activity of RT and CT in malignant cells but not normalcells.

In addition, the present invention discloses the surprising discoverythat benzoic acid derivatives that inhibit tyrosinase, and hence,melanogenesis, can potentiate RT and CT activities in melanotic as wellas non-melanin expressing carcinoma cells.

REFERENCES CITED

Numerous references, including patents, patent applications and variouspublications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entirety andto the same extent as if each reference was individually incorporated byreference.

1. A method of treating cancer in a mammal, comprising administering toa mammal in need of such treatment a therapeutically effective amount ofa benzoic acid derivative, excluding para-amino-benzoic acid (PABA) 2.The method of claim 1, wherein the treatment comprises administering atleast one of chemotherapy or radiation therapy to said mammal.
 5. Themethod of claim 1, which comprises administering a benzoic acidderivative which inhibits tyrosinase.
 4. The method of claim 1, whichcomprises administering a benzoic acid derivative selected from thecompounds depicted in FIG.
 1. 5. The method of claim 4, wherein thebenzoic acid derivative is C-45 and has the following structure:


6. The method of claim 1, wherein the cancer is selected from the groupconsisting of melanoma, breast carcinioma and lung carcinoma.
 7. Themethod of claim 6, wherein the mammal is a human.
 8. The method of claim7, wherein the cancer is melanoma.
 9. The method of claim 7 wherein thecancer is breast carcinoma.
 10. The method of claim 7, wherein thecancer is lung carcinoma.
 11. The method of claim 2, wherein the canceris selected from the group consisting of melanoma, breast carcinioma andlung carcinoma.
 12. The method of claim 11, wherein the benzoic acidderivative is administered in combination with an effective amount ofpaclitaxel.
 13. The method of claim 11, wherein the benzoic acidderivative is administered in combination with radiation therapy.
 14. Amethod of screening for lead compounds that bind to the PXR/SXRreceptor, comprising i) transfecting host cells with a reporterconstruct carrying a xenobiotic response element positioned upstream ofa reporter gene; ii) contacting the cells with a test compound; iii)measuring the change in reporter gene expression, wherein increasedreporter gene expression indicates binding and activation of thexenobiotic response element; and iv) selecting as a lead compound a testcompound which increases reporter gene expression compared to anuntreated control.
 15. The method of claim 14, wherein the xenobioticresponse element comprises the nucleotide sequence 5′-AGTTCA-3′ (SEQ IDNO: 1).
 16. A method of treating cancer in a mammal comprisingadministering to a mammal in need of such treatment an effective amountof a compound that binds to and activates the PXR/SXR receptor, furtherin combination with radiation therapy or chemotherapy, wherein thecancer comprises cells that express the PXR/SXR receptor.
 17. The methodof claim 16, wherein the lead compound is a benzoic acid derivative. 18.The method of claim 17, wherein the benzoic acid derivative is selectedfrom the group consisting of the compounds depicted in FIG.
 1. 19. Themethod of claim 17, wherein the compound decreases the expression levelof a DNA repair protein and increases the level of a cell cycleprogression protein.
 20. The method of claim 17, wherein the DNA repairprotein or cell cycle progression protein is selected from the groupconsisting of BRCA-2, Id-1, Id-2 and Id-3 and the cell cycle progressionprotein is selected from the group consisting of CDC25A, CDC2, Wee-1,Myt-1, cyclin A, cyclin B, CHK 1 and CHK2 and LATS1.
 21. The method ofclaim 19, wherein the DNA repair protein is BRCA-2 and the cell cycleprogression protein is CDC25A.
 22. The method of claim 15, wherein themammal is a human.
 23. The method of claim 17 wherein the compoundincreases apoptosis of the cancer cells.
 24. The method of claim 16,wherein the cancer is melanoma.